6 Enzymes (DIY)

• Enzyme (CHARACTERISTIC - 1)

  1. Enzymes are mostly globular proteins

  • Consist of 1 or more polypeptide chains folded to form a globular unit (tertiary or quaternary structure)
  • Primary structure determines its secondary and tertiary structure
  • Specifies the overall 3D conformation/ structure of the enzyme
  • Action of enzymes depends on their 3D conformation
  • Exceptions: some enzymes can be composed of RNA - ribosome/ complexes of RNA and protein
• Enzymes (CHARACTERISTICS - 2)

  1. Enzymes increase the rate of reaction

  • Rate of enzyme-catalyse reactions are typically 10^6 to 10^12 times greater than those uncatalysed
• Enzymes (CHARACTERISTICS - 3)

  1. Enzymes operate at milder reaction conditions

  • Temperatures below 100degC, normal atmospheric pressure and nearly neutral pH normally encountered in organisms
  • VS chemical catalysts that require elevated temperatures and pressures and extreme pH to be effective
• Enzymes (CHARACTERISTIC - 4)

  1. Enzymes exhibit substrate specificity

  • Usually catalyses a specific chemical reaction
  • Absolute specificity (enzyme catalyses a specific reaction)
  • Group specificity (enzyme acts on one type of chemical bond in a variety of substances)
• Active Site

  • Only a small region of the enzyme binds with the substrate (ACTIVE SITE)
  • precise 3D groove of the enzyme at the active site which gives it a specific 3D conformation
  • specificity is attributed to the complementary conformation and charge between substrate and active site
  • NOT rigid → structure changes as substrate enters so the active site fits more snugly around the substrate to form a more stable structure
  • Typically consists of 3-12 amino acids
• Amino Acid Residues (1 - Contact Residues)

  1. Contact residues

  • bind reversible with substrate to position it in the correct orientation
  • substrate is held in the active site by weak interactions (hydrogen bonds, ionic bonds, hydrophobic interactions)
  • RESPONSIBLE for enzyme specificity 
• Amino Acid Residues (2 - Catalytic Residues)

  1. Catalytic residues

  • act on the bonds in substrate molecule
  • side chains/ R-groups of a few amino acid residues catalyse the conversion of the substrate to product
• Amino Acid Residues (3 - Structural Residues)

  1. Structural residues

  • interacts to maintain the overall 3D conformation of the protein for proper functioning of the protein
• Amino Acid Residues (4 - Non-essential residues)

  1. Non-essential residues

  • generally found on the surface of the protein
  • no specific functions
• "Lock and Key" Hypothesis

  Enzymes have a specific active site conformation and charge produced by the 3-dimensional folding of the polypeptide chain
• Active site

  Specific surface conformation and charge of an enzyme
• Substrate


  “Key” conformation and charge are complementary to the enzyme active site (“lock”)
• The basis of specificity is the complementary nature of the enzyme and substrate
• Enzyme-substrate interaction

  1. Collision in correct orientation

  2. Substrate fits into active site

  3. Temporary enzyme-substrate (ES) complex forms

  4. Catalysis occurs

  5. Products formed
• Products formed

  No longer fit into the active site and are released into the surrounding medium
• The active site is free to receive further substrate molecules after products are released
• The enzyme and its active site are not altered at the end of the reaction
• Induced-Fit Hypothesis

  The active site of enzymes is generally complementary in conformation, but not a perfect fit to its substrate
• Induced-Fit Mechanism

  1. Substrate binds

  2. Induces change in enzyme conformation

  3. Allows for more precise fit

  4. Enzyme performs catalytic function more effectively
• Induced change in shape of enzyme

  • More snug fit = more weak bonds formed
  • Active groups brought to the right location
• The 3D conformation of enzyme reverts to its original state upon completion of the reaction and release of the product molecules
• Lowering of Activation Energy Barriers

  • Activation Energy: initial investment of energy for a reaction to proceed
  • enzymes speed up the reaction by lowering the activation barrier of the reaction
  • enables more reactant molecules to reach the transition state at moderate temperatures
  • does so by 5 different mechanisms
• MECHANISM of lowering Ea barrier (1 - Proximity Effects)

  1. Proximity Effects

  • temporary binding of reactants next to each other in enzyme active site → increases chance of reaction
  • uncatalysed reactions instead depend on random collisions between reactant molecules
• MECHANISMS of lowering Ea barrier (2 - Strain Effects)

  1. Strain effects

  • slight distortion of the reactants as they bind to the enzyme → strains bonds which are to be broken → increases chance of breakage
• MECHANISMS of Ea barrier (3 - Orientation Effects)

  1. Orientation effects

  • reactant are held by the enzyme in a way where bonds are exposed to chemical reactions
• MECHANISMS of Ea barrier (4 - Microenvironment Effects)

  1. Microenvironment effects

  • hydrophobic amino acids create a “water-free” zone in which non-polar reactants may react more easily
• MECHANISMS of Ea barrier (5 - Acid-base catalysis)

  1. Acid base catalysis

  • acidic and basic amino acids in the enzyme facilitate catalysis
• Temperature (RATE FACTOR - 1)

  1. Temperature < Optimum Temperature

  • Increase in temperature → increase in kinetic energy of enzyme and substrate molecules → increases the number of molecules having sufficient energy to overcome the activation energy barrier to form the products of the reaction
  • Increase in frequency of effective collisions between substrate and enzyme active site → increase in rate of formation of enzyme-substrate complexes
  • Q10 (Temperature Coefficient) = factor by which rate increases with each 10degC rise in temperature
• Temperature (RATE FACTORS - 2)

  1. Optimum temperature

  • Reaction rate increases with temperature only until optimal temperature is reached
  • Each enzyme has an optimal temperature at which the rate of enzyme reaction proceeds at a maximum rate (humans: 25 - 40degC)
  • Some enzymes have a higher optimum temperature
  • These tend to have a higher proportion of disulfide bonds (strong covalent bonds) or numerous intramolecular interactions that hold the tertiary structure of the enzyme together
• Temperature (RATE FACTORS - 3)

  • Kinetic energy of enzyme and substrate molecules continues to increase with increasing temperature, frequency of effective collisions between substrate and enzyme active site decreases due to denaturation
  • At high temperatures, intramolecular vibrations increases - hydrogen bonds, ionic bonds and hydrophobic interactions that stabilise the active site conformation are broken - denaturation
  • Active site conformation is lost → no longer complementary to the substrate
  • Rate of formation of enzyme-substrate complexes decreases → decrease in the rate of reaction
• pH (RATE FRACTOR - 1)

  • Each enzyme has an optimal pH at which it is most active → rate of reaction is maximum here
  • Deviation from the optimum pH → lowering of the rate of reaction
  • Excess [H+] or [OH-] ions will neutralise negatively and positively charged R-groups of amino acids in the enzyme respectively
  • Excess H+ results in -COO- groups becoming -COOH
  • Excess -OH- results in -NH3+ becoming -NH2
• pH (RATE FACTOR - 2)

  • If neutralised R groups belong to:
  • Structural amino acid residues → disruption of ionic bonds and hydrogen bonds which determine the tertiary structure of the protein → changes specific 3D conformation of the enzyme active site → enzyme is denatured
  • Contact amino acid residues → substrate may not be able to bind to the enzyme active site to form enzyme-substrate complex
• pH (RATE FACTOR - 3)

  • If neutralised R groups belong to:
  • Catalytic amino acid residues in the active site → catalysis will not take place
  • Part of the protein substrate → charges on its residues will change → affects substrate interaction with the enzyme active site and catalysis
• Enzyme Concentration (RATE FACTOR -1)

  • Rate of enzyme-controlled reaction is dependent on the frequency of effective collisions between enzyme molecules and substrate molecules
  • Increased enzyme concentration
  • More active sites available per unit volume for substrates to bind to → frequency of effective collisions between enzyme molecules and substrate molecules increases → increased rate of formation of enzyme-substrate complexes → reaction rate increases
• Enzyme Concentration (RATE FACTOR - 2)

  • At linear portion of graph → enzyme concentration is limiting
  • Any increase in enzyme concentration will result in a proportional increase in rate of reaction
  • At curved portion of graph → enzyme concentration is not the only limiting factor
  • Some other factor is also limiting
  • At the plateau → enzyme concentration is no longer the limiting factor
  • Other factors are limiting the rate of reaction 
  • Increasing enzyme concentration no longer increases the rate of reaction

  
• Substrate Concentration (RATE FACTOR -1)

  • At linear portion of graph
  • Rate of reaction increases proportionally with an increase in substrate concentration
  • Frequency of effective collisions between enzyme and substrate molecules increases → rate of enzyme-substrate complex formation increases → rate of reaction increases
  • At low substrate concentration, the substrate concentration is limiting
  • There are more active sites of the enzymes available to catalyse the reaction and the limited supply of substrate molecules largely determines the rate of reaction
• Substrate Concentration(RATE FACTOR - 2)

  • At curved portion of graph
  • Enzyme active sites start to get saturated and limits the rate of reaction
  • At plateau portion of graph
  • The rate of reaction has reached its maximum velocity (Vmax)
  • Enzyme saturation is reached whereby all available active sites are occupied by substrate molecules
  • Substrate concentration is no longer limiting → further increases in substrate concentration will not cause the rate of reaction to increase further
  • Enzyme concentration is limiting
• Substrate Concentration (RATE FACTOR - 3)

  • Michaelis constant (Km) = the concentration of substrate which allows the reaction to attain half its maximum rate (½ Vmax)
  • Always the same for a particular enzyme, but varies from one enzyme to the other
  • An inverse measurement of the affinity of the enzyme for its substrate (tendency of enzyme to bind to substrate)
  • Low Km: low [S], high affinity between enzyme and substrate
  • High Km: high [S], low affinity between enzyme and substrate
• Enzyme Co-factors (RATE FACTOR - inorganic cofactors)

  • Cofactors: Some enzymes require additional non-protein substances for catalytic activity
  • Inorganic ions
  • Many enzymes require certain metal ions to change non-functioning active site to a functioning one
  • Some common cofactors: Ca2+, Mg2+, Mn2+, Cu2+ and Zn2+
  • The attachment of the ion with the main enzyme (apoenzyme) changes the shape of the enzyme so as to allow the enzyme-substrate complex to form more easily
• Enzyme Co-factor (RATE FACTOR - organic cofactor)

  1. Organic cofactors

  • Coenzymes
  • Usually bind loosely and briefly to enzymes
  • Prosthetic group
  • Permanently bound to enzymes via strong covalent bonds